先天性肛门直肠畸形患儿血清VA及肠组织RARα表达及意义

Expression and significance of serum content of vitamin A and intestinal expression of retinoic acid receptor α in children with congenital anorectal malformation

目的检测先天性肛门直肠畸形(congenital anorectal malformation,ARM)患儿血清维生素A(vitamin A,VA)水平及肠组织维甲酸受体α(retinoic acid receptorα,RARα)的表达特点,探讨其与ARM肠神经系统(enteric nervous system,ENS)异常的可能联系。方法利用高效液相色谱法检测32例初诊ARM新生儿血清VA浓度,选取30例非消化道畸形新生儿血清作为对照;收集30例ARM患儿直肠末端及造瘘口横结肠组织,选取4例非消化道畸形患儿直肠组织作为对照,RT-PCR检测各组肠组织RARαmRNA表达水平;根据患儿直肠末端病理检查结果将直肠组织分为神经节细胞发育正常组和异常组(n=6),免疫组织化学染色和Western blot检测两组直肠RARα蛋白的表达水平。结果 ARM新生儿血清VA平均浓度显著低于对照组(0.40±0.14μmo L/L vs 0.54±0.10μmo L/L,P<0.01);ARM患儿直肠末端RARαmRNA表达水平(0.78±0.40)显著低于造瘘口横结肠(1.28±0.72,P<0.01)和正常对照组直肠组织(1.12±0.14,P<0.01),而造瘘口横结肠与正常对照组直肠无显著性差异(P>0.05);RARα蛋白在直肠神经纤维和神经节细胞核呈阳性表达,神经节细胞发育异常组直肠RARα蛋白表达水平显著低于正常组(0.77±0.08 vs 1.03±0.06,P<0.01)。结论 ARM患儿存在血清VA及直肠组织RARα表达异常。

Objective To investigate the serum content of vitamin A（ VA） and the intestinal expression of retinoic acid receptor α（ RARα） in the children with congenital anorectal malformation（ ARM）,and explore the possible association of them with abnormalities of ARM enteric nervous system（ ENS）. Methods A total of 32 freshly-diagnosed ARM neonates who undergoing elective staged surgical treatment in our hospital from February 2015 to February 2017 were recruited in this study. High performance liquid chromatography was used to detect the serum VA content in the 32 ARM neonates and 30 neonates（ as control） without alimentary tract malformations. RT-PCR was employed to detect the mRNA expression of RARα in intestinal specimens at the end of rectum and stoma from 30 ARM neonates and 4 neonates（ as control） without alimentary tract malformations. According to the results of pathological study on the intestinal tissues at the end of rectum,the tissues were divided into normal and abnormal ganglion cells groups（ n = 6 for each group）. Then immunohistochemical assay and Western blotting were adopted to detect the expression of RARα protein in the rectum specimens. Results The serum content of VA was significantly lower in the ARM neonates than the control neonates（ 0. 40 ± 0. 14 vs 0. 54 ± 0. 10 μmol/L,P〈0. 01）. The expression level of RARα mRNA was significantly lower in the rectum of ARM（ 0. 78 ± 0. 40） than in the colon of stoma（ 1. 28 ± 0. 72,P〈0. 01） and the rectum of control（ 1. 12 ± 0. 14,P〈0. 01）,but there was no difference in the level between the colon of stoma and rectum of control（ P〉0. 05）. RARα were expressed in the nucleus of ganglion cells and nerve fibers in the rectum. The protein level of RARα was significantly lower in the abnormal ganglion cells group than the normal ganglion cells group（ 0. 77 ± 0. 08 vs 1. 03 ± 0. 06,P〈0. 01）.Conclusion ARM neonates have lower serum VA content and abnormal intestinal expression of RARα.